1. Field of the Invention
The present invention relates to compositions and methods useful for detecting the presence and quantifying the amount of double-stranded DNA-activated protein kinase (DNA-PK) in a biological sample and a method of monitoring DNA-PK activity or DNA-PK activity state in living cells.
2. Background of the Related Art
Eukaryotic cells contain many different protein kinases, each having specific functions and properties. Protein kinases are important signal transduction enzymes that regulate many aspects of cell metabolism and cell growth in eukaryotic cells. Protein kinases alter the properties of other enzymes or structural proteins by transferring a phosphate from a donor molecule, often ATP, to one or more acceptor amino acids of a substrate protein. Although the basic amino acids lysine, arginine and histidine can be phosphorylated by certain protein kinases which are primarily associated with bacterial organisms, the most common and more widely recognized phosphate acceptor amino acids in protein substrates in eukaryotic organisms are serine, threonine and/or tyrosine residues. The most common and more well-studied protein kinases are either protein-serine/threonine kinases, which are capable of transferring phosphate groups from donor molecules to serine and threonine acceptor amino acid residues in substrate proteins, or protein-tyrosine kinases, which are capable of transferring phosphate groups from donor molecules to tyrosine acceptor amino acid residues in substrate proteins.
Protein kinases have been found to modulate the activities of proteins in cells by phosphorylating their specific protein substrates. These specific phosphorylation reactions constitute a major mechanism for regulating biochemical pathways in multicellular eukaryotic organisms. Nuclear events, metabolic processes and signal transduction at the cytoplasmic membrane are coordinated through the phosphorylation and dephosphorylation of proteins that perform and, control these processes. Transcription and DNA replication are also regulated by phosphorylation. Most replication and transcription factors are phosphorylated at several sites and often by several different protein kinases. Kinases have been described that are activated by a variety of agents including cyclic nucleotides, phorbol esters, phospholipids, calcium ions, heme groups, double-stranded RNA (dsRNA); and double-stranded DNA (dsDNA). A growing number of nuclear regulatory proteins are known to be phosphorylated, but the kinases which phosphorylate these proteins frequently remain unidentified. Furthermore, many of the kinases which have been identified are not well characterized.
The characterization of many protein kinases has been facilitated by the development of in vitro protein kinase assays that specifically and quantitatively detect a particular kinase in a biological sample. Early work in this area was performed by Glass, et al. and is reported in Anal. Biochem., 87:566 (1978). Glass, et al. developed a procedure for isolating phosphorylated peptides and proteins on ion exchange papers under acidic conditions that is applicable to the study of protein-serine/threonine and protein-tyrosine kinases. The method is not applicable to the study of protein kinases that phosphorylate the basic amino acids because the bond between the phosphate group and the basic amino acid is hydrolyzed in the acidic conditions of the method. The paper-binding method was applied to protein kinase, assays that utilized synthetic peptides with amino acid sequences corresponding to the primary amino acid sequence at the phosphorylation sites of native proteins. Synthetic peptide substrates and methods for their rapid separation from kinase assay reaction mixtures have been useful in the detection, quantitation and characterization of numerous protein kinases.
Studies using substrate proteins and synthetic peptide substrates enabled the demonstration that a given protein kinase, whether of the protein-serine/threonine or the protein-tyrosine kinase class, did not phosphorylate all acceptor amino acid residues in a protein or peptide substrate, but was capable of specific selection of the serine, threonine or tyrosine that was to be phosphorylated in its various protein and peptide substrates. These studies lead to the determination that each protein kinase exhibited a specific phosphorylation site consensus sequence motif requirement for selection of the acceptor amino acid (phosphorylation site) in protein substrates or synthetic peptide substrates. Phosphorylation site consensus sequence motifs are comprised of a phosphorylation site acceptor amino acid (serine, threonine or tyrosine) embedded in a sequence or arrangement of amino acids that is specifically recognized by the protein kinase such that the kinase transfers the phosphate to that acceptor amino acid rather than to serines, threonines or tyrosines that are found elsewhere in the substrate but which are not so embedded. Pearson, et al. in Methods Enzymol., 220:62 (1991) tabulated protein kinase phosphorylation site consensus sequence motifs for over 240 protein-serine/threonine and protein-tyrosine kinases. That tabulation was derived from studies of protein kinases utilizing assays containing protein substrates or assays containing synthetic peptide substrates that were synthesized as analogs of natural phosphorylation site sequences.
Synthetic peptide analogs of phosphorylation site consensus sequence motifs are useful for detecting, quantifying and characterizing protein kinases. Synthetic peptide substrates have played an important role in the study of protein kinase (PK) substrate specificity as well as in the measurement of protein kinase activities in cell extracts. The major goals of designing synthetic peptide substrates for protein kinases are to construct peptides that have excellent kinetic properties and a high degree of specificity (Kemp, et al, 1991, Methods Enzymol., 200:121; Pearson, et al., 1991, Methods Enzymol., 200:62).
While synthetic peptides with amino acid sequences corresponding to the primary amino acid sequence at the phosphorylated sties of proteins have been found to serve as specific substrates for certain protein kinases, the degree of specificity of synthetic peptide substrates varies widely. A number of synthetic peptides act as substrates for multiple protein kinases. For example, phosphorylate kinase, protein kinase C, and the multi-functional calmodulin-dependent protein kinase all phosphorylate Ser 7 in a glycogen synthase peptide.
Synthetic peptide substrates for protein kinases are particularly useful if they are specifically phosphorylated only by the protein kinase of interest. A specific synthetic peptide substrate comprises a kinase-specific phosphorylation site consensus sequence motif and sufficient additional amino acids (amino acid spacer sequences) so as to create a peptide providing excellent kinetic properties in an assay. Preferably, a specific synthetic peptide substrate provides a kinase-specific phosphorylation site consensus sequence motif and sufficient amino acid spacer sequences so as to provide excellent kinetic properties in an assay and which amino acid spacer sequences do not provide another phosphorylation site consensus sequence motif. Additionally preferable is a specific synthetic peptide substrate providing a kinase-specific phosphorylation site consensus sequence motif, sufficient amino acid spacer sequences to provide excellent kinetic properties in an assay that do not provide another phosphorylation site consensus sequence motif and which amino acid spacer sequences do not contain another phosphate acceptor amino acid. To provide for economic synthetic procedures, it is especially preferable that a specific synthetic peptide substrate be as short as possible while providing the features described above.
Recently, a kinase that undergoes activation by linear double stranded (ds) DNA was discovered. This kinase is now known as the double-stranded DNA-activated (or dependent) protein kinase (DNA-PK). DNA-PK is perhaps the most abundant nuclear protein kinase in human cells. It is active in vitro only when certain double-stranded DNA molecules are also present. Both natural DNAs and synthetic oligonucleotides are DNA-PK activators. DNA-PK is a protein-serine/threonine kinase and therefore phosphorylates certain serine and threonine residues in specific polypeptide sequences by transferring a phosphate from the xcex3-position of a suitable phosphate donor, such as adenosine triphosphate (ATP), to specific serine and threonine acceptor amino acids in its substrate proteins.
DNA-PK phosphorylates several viral and cellular proteins in vitro, including the simian virus 40 large tumor antigen (SV40 TAg), heat shock protein (hsp90), the human and murine p53 tumor suppressor proteins, and several transcription factors including Sp1, Oct-1, Fos, Jun and Serum Response Factor (SRF). Many of these proteins are DNA-binding proteins that function in RNA transcription, DNA replication, DNA recombination and DNA repair. The ability of DNA-PK to phosphorylate these regulatory proteins provides a means for elucidating important regulatory pathways. DNA-PK may be useful for studying the extent to which phosphorylation regulates gene expression and cell growth. Additionally, DNA-PK may be an important cellular enzyme in that it may play a role in transcription regulation, DNA replication, and/or DNA repair. DNA-PK may be a regulatory kinase which may be useful in detecting damaged DNA. The phosphorylation of cellular protein substrates by DNA-PK may activate cell cycle checkpoint mechanisms that arrest cell cycle progression in response to DNA damage. DNA-PK may also regulate processes involved in the development of cellular immunity. These and other properties suggest that DNA-PK may be extremely important for regulating the state or utilization of cellular DNA, especially in primate cells.
To determine the function of DNA-PK, it is necessary to understand the factors that result in its activation in cells. A significant technical difficulty in characterizing and studying DNA-PK has been the lack of a simple, quantitative, specific assay analogous to the synthetic peptide substrate assays, as mentioned above, which are available for other protein-serine/threonine kinases and protein-tyrosine kinases. The procedures for the synthesis of peptides for the study of cyclic nucleotide-dependent protein kinases may be applicable to the preparation of synthetic peptides which are specific for DNA-PK.
Previous research has shown that purified SV40 TAg and murine p53 protein were phosphorylated in vitro on serines by the human DNA-activated protein kinase, DNA-PK (Lees-Miller, et al., 1990, Mol. Cell Biol., 10:6472-6481). More recently it was shown that the 14-residue synthetic peptide corresponding to TAg Thr661 to Pro674 was phosphorylated by DNA-PK primarily on the serine equivalent to TAg residue 665 (Chen, et al., 1991, J. Virol., 10:5131-5140).
Several attempts have been made to provide a quantitative specific assay for DNA-PK. However, these attempts have failed. All known natural protein substrates for DNA-PK are also phosphorylated by other protein kinases. For example, casein which is a substrate of DNA-PK, is inexpensive and readily available. Unfortunately, casein is phosphorylated by several protein-serine/threonine kinases and therefore its phosphorylation, after incubation with a biological sample, for example, would not be a specific measure of DNA-PK activity in the biological sample. Hsp90 is a more specific DNA-PK substrate, but it can also be phosphorylated by CKII (and perhaps other kinases). In addition, although hsp90 is abundant and easy to purify, its purification requires several days"" work. Further, the quantitation of hsp90 phosphorylation is cumbersome and the kinetic constants for hsp90 phosphorylation are less than ideal. Consequently, simple cost-effective methods for accurately detecting the presence of DNA-PK are needed.
A further complication in characterizing and studying DNA-PK has been the lack of a meaningful correlation between the activity observed in vitro and DNA-PK activity in vivo. DNA-PK is activated in vitro by linear double-stranded DNA fragments, by nicked or gapped double-stranded DNAs or by DNAs with single-to-double strand transitions (bubbles, forks, hairpins, etc.) (Morozov, et al., 1994, J. Biol. Chem. 269:16684-16688). The preparation of cell extracts, for the determination of DNA-PK activity, inevitably introduces nicks and breaks into endogenous cellular DNA. DNA-PK or its Ku targeting/regulatory domain binds to the DNA at these nicks or breaks and becomes artificially activated. This artificial activation of DNA-PK upon cell disruption to form cell extracts makes it impossible to measure the status of intracellular DNA-PK activity using an in vitro cell extract assay.
An intracellular, in vivo method for measuring the intracellular DNA-PK activity and the activation status of DNA-PK within, a cell would provide an alternative strategy. Such a strategy could include a determination of the in vivo phosphorylation state of known substrates. However, nearly all of the putative physiological substrates of DNA-PK are proteins that are present in cells at low abundance, making quantitation difficult. In addition, these proteins are also phosphorylated by other protein kinases. Consequently, it has not been possible to ascertain the activity state of DNA-PK in living cells by examining the phosphorylation state of suspected substrates. Further, determining the activity of DNA-PK in cells or changes in its activity by examining the state of endogenous substrates is also complicated by the fact that determining the phosphorylation state of these proteins most often requires incubating cells with radioactive 32PO4. The radiation emitted by this compound produces single-strand and double-strand breaks in the endogenous cellular DNA which alters the activity of DNA-PK in living cells. In fact, as previously mentioned, part of the in vivo function of DNA-PK may be to detect such DNA damage. It is known, for example, that radioactive nucleic acid precursors (e.g. [3H]-thymidine) cause an accumulation of the tumor suppressor protein p53, a putative DNA-PK substrate (Dover, et al.,1994, J. Cell Sci. 107:1181-1184). Further, p53 accumulation in response to DNA breaks and other damage arrests cell cycle progression in the G1 phase of the cell cycle (Nelson, et al., 1994, Mol. Cell. Biol. 14:1815-1823).
In light of the foregoing, it is a purpose of the present invention to provide a composition that comprises a synthetic peptide substrate which is a specific substrate for DNA-PK and which therefore can be used to specifically detect, quantitate and monitor DNA-PK activity.
Another purpose of the present invention is to provide specific quantitative methods of detecting the presence of DNA-PK activity in a biological sample.
Another purpose of the present invention is to provide methods of monitoring DNA-PK activity in living cells.
A further goal of the present invention is to provide a method of identifying agents which modulate DNA-PK activity in vivo and in vitro.
A further purpose of the present invention is to provide a kit for in vitro detection of the presence and relative amount of DNA-PK in a biological sample.
An additional purpose of the present invention is to provide a kit for monitoring the intracellular activity and activation status of DNA-PK.
These and other purposes and goals are accomplished by the present invention which provides a composition that comprises a synthetic peptide substrate that is a specific substrate for DNA-PK and which therefore is useful to specifically detect, quantitate and monitor DNA-PK activity. The synthetic peptide substrate of the present invention is a peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor.
Synthetic peptide substrates providing some or all of these features include: Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu (SEQ ID NO: 1), Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro Leu Ser Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro Pro (SEQ ID NO: 2), Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro Tyr Lys Lys (SEQ ID NO: 3), Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Lys (SEQ ID NO: 4), Asn Asn Val Leu: Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Lys Lys (SEQ ID NO: 6), Met Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg (SEQ ID NO: 7), Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Lys (SEQ ID NO: 8), Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 11), Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Leu Lys Lys (SEQ ID NO: 12), Glu Pro Pro Gln Ser Leu Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 14), Glu Pro Pro Gln Ser Gln Gln Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 15), Glu Pro Pro Leu Thr Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 16), Glu Pro Pro Asp Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 17), Pro Glu Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 18) and Pro Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 19).
In a preferred embodiment the synthetic peptide substrate providing all of the features described above to provide specific recognition and phosphorylation by DNA-PK is a synthetic peptide substrate which is identical to or a variant of the amino acid sequence found at the amino terminus of human or murine p53 tumor suppressor proteins, and which contains either Ser15 of human p53 tumor suppressor protein or Ser7 or Ser18 of murine p53 protein, including the following synthetic peptides: Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu (SEQ ID NO: 1), Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro Leu Ser Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro Pro (SEQ ID NO: 2), Met Glu Glu Ser Gln Ser Asp Ile Ser. Leu Glu Leu Pro Tyr Lys Lys (SEQ ID NO: 3), Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Lys (SEQ ID NO: 4), Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Lys (SEQ ID NO: 8), Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 11), Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Leu Lys Lys (SEQ ID NO: 12), Glu Pro Pro Gln Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 15), Glu Pro Pro Asp Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 17), Pro Glu Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 18), Pro Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 19), Pro Leu Ser Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro Pro-Lys Lys (SEQ ID NO: 63), and Pro Leu Ser Gln Glu Ala Phe Ala Gly Leu Trp Lys Leu Leu Pro Pro-Lys Lys (SEQ ID NO: 64).
In a most preferred embodiment the synthetic peptide substrate providing all of the features described above to provide specific recognition and phosphorylation by DNA-PK is a synthetic peptide substrate which is a variant of the amino acid sequence found at the amino terminus of human p53 tumor suppressor proteins, and which contains Ser15 of human p53 tumor suppressor protein, including the following peptides: Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 11), Glu Pro Pro Gln Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 15), Pro Glu Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 18) and Pro Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 19).
The present invention also provides a quantitative method for detecting the presence of DNA-PK activity in biological samples. The method includes forming a reaction mixture by contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate in the presence or absence of added exogenous DNA. The synthetic peptide substrate is a peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutaminie-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. Synthetic peptide substrates providing some or all of these features include: Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu (SEQ ID NO: 1), Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro Leu Ser Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro Pro (SEQ ID NO: 2), Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro Tyr Lys Lys (SEQ ID NO: 3), Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Lys (SEQ ID NO: 4), Asn Asn Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Lys Lys (SEQ ID NO: 6), Met Ala lie Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg (SEQ ID NO: 7), Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Lys (SEQ ID NO: 8), Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 11), Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Leu Lys Lys (SEQ ID NO: 12), Glu Pro Pro Gln Ser Leu Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 14), Glu Pro Pro Gln Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 15), Glu Pro Pro Leu Thr Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 16), Glu Pro Pro Asp Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 17), Pro Glu Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 18) and Pro Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 19).
In a preferred embodiment of this method, a biological sample is contacted with a synthetic peptide substrate which provides all of the features described above to provide specific recognition and phosphorylation by DNA-PK and which is identical to or a variant of the amino acid sequence found at the amino terminus of human or murine p53 tumor suppressor proteins, and which contains either Ser15 of human p53 tumor suppressor protein or Ser7 or Ser18 of murine p53 protein, including the following synthetic peptides: Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu (SEQ ID NO: 1), Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro Leu Ser Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro Pro (SEQ ID NO: 2), Met Glu Glu Ser Gln Ser Asp Ile Ser Leu Glu Leu Pro Tyr Lys Lys (SEQ ID NO: 3), Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Lys (SEQ ID NO: 4), Glu Pro Pro Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Lys (SEQ ID NO: 8), Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 11), Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Leu Lys Lys (SEQ ID NO: 12), Glu Pro Pro Gln Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 15), Glu Pro Pro Asp Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 17), Pro Glu Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 18), Pro Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 19), Pro Leu Ser Gln Glu Thr Phe Ser Gly Leu Trp Lys Leu Leu Pro Pro-Lys Lys (SEQ ID NO: 63), and Pro Leu Ser Gln Glu Ala Phe Ala Gly Leu Trp Lys Leu Leu Pro Pro-Lys Lys (SEQ ID NO: 64).
In a most preferred embodiment of this method, a biological sample is contacted with a synthetic peptide substrate which provides all of the features described above to provide specific recognition and phosphorylation by DNA-PK and which is a variant of amino acid sequence found at the amino terminus of human p53 tumor suppressor proteins, and which contains Ser15 of human p53 tumor suppressor protein, including the following peptides: Glu Pro Pro Leu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 11), Glu Pro Pro Gln Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 15), Pro Glu Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 18) and Pro Glu Ser Gln Glu Ala Phe Ala Asp Leu Trp Lys Lys (SEQ ID NO: 19).
Next, the reaction mixture is incubated for a time at a temperature to allow the transfer of phosphate from the phosphate donor to the acceptor amino acid in the synthetic peptide substrate. After the incubation step, the transfer of phosphate from the phosphate donor to the acceptor amino acid in the synthetic peptide substrate is stopped. Finally, the method is used to determine the amount of phosphate which was transferred from the phosphate donor to the acceptor amino acid in the synthetic peptide substrate and to correlate the amount of phosphate transferred with a concentration of DNA-PK in a biological sample.
In a preferred embodiment of the invention, the gamma phosphate of the phosphate donor, ATP, contains a radioactive phosphorous isotope, e.g. [32P] or [33P]. After the incubation, the synthetic peptide, containing the radioactive phosphate transferred by DNA-PK from the radiolabeled ATP, is separated from remaining labeled ATP and phosphate using the tag moiety of the peptide substrate. In a preferred embodiment, this separation is accomplished by absorbing the peptide to phosphocellulose and washing the phosphocellulose with dilute acid to remove the unreacted ATP phosphate donor. The amount of radioactivity transferred to the peptide is then determined using a device, such as a scintillation counter, a beta particle detector, gamma ray detector, or a phosphoimager, to quantitate the radioactivity in a known portion of the reaction. The amount of phosphate transferred to the peptide is then calculated from the known specific activity of the ATP.
A method of detecting the presence of linear double-stranded DNA in a biological sample is also provided. The method includes forming a reaction mixture by contacting a biological sample with a detectably-labeled phosphate donor, DNA-PK and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1),a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic: acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. The transfer of phosphate from the phosphate donor to the peptide substrate is indicative of the presence of linear double-stranded DNA in the sample because it is required for activation of DNA-PK and was not supplied in the reaction mixture.
A method of detecting the presence of substances in a sample that alter the activity of DNA-PK and DNA is also provided. The method includes forming a reaction mixture by contacting increasing amounts of a sample with a detectably-labeled phosphate donor, DNA-PK, linear double-stranded DNA and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair arid forming an amino acid pair-enhancer unit; (3) a, first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. Changes in the amount or rate of transfer of phosphate from the phosphate donor to the peptide substrate caused by contacting the sample is related to the presence of substances that alter the activity of DNA-PK.
The present invention also provides a method of detecting the presence of protein phosphatases in a biological sample. The method includes contacting a biological sample with a phosphorylated synthetic peptide substrate defined by the following features to provide specific recognition by DNA-PK: (1) a phosphorylated amino acid pair which may include phosphoserine-glutamine (PO4xe2x80xa2Ser-Gln), phosphothreonine-glutamine (PO4xe2x80xa2Thr-Gln), glutamine-phosphoserine (Gln-PO4xe2x80xa2Ser), or glutamine-phosphothreonine (Gln-PO4xe2x80xa2Thr); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. The loss of phosphate from the phosphorylated peptide substrate is related to the presence of protein phosphatase in the sample.
The present invention also provides a composition useful in the specific, quantitative detection of DNA-PK activity in a biological sample. The composition is a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamne-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor.
The present invention also provides a kit for detecting the presence of DNA-PK activity in a biological sample for use in accordance with the method of the present invention. The kit includes a detectably-labeled phosphate donor, double-stranded DNA, a composition useful in specific detection and quantitation of DNA-PK which comprises a synthetic peptide substrate that is phosphorylated by DNA-PK, a negative control peptide that is not phosphorylated by DNA-PK, a preparation of DNA-PK and a reagent to separate the substrate peptide from the phosphate donor.
A negative control peptide that is not phosphorylated by DNA-PK may include a synthetic peptide with an amino acid sequence and composition similar to that of the synthetic peptide substrate, but with a variation of:the phosphorylation site consensus sequence motif such that the acceptor amino acid is no longer phosphorylated by DNA-PK. Peptides with amino acid sequences and compositions that are similar to that of the synthetic peptide substrate may generally be comprised of the same amino acids as the peptide substrate with an alteration of the primary sequence such that the peptide no longer contains the appropriate phosphorylation site consensus motif and is thus not phosphorylated by the DNA-PK. Such negative control peptides thus provide non-phosphorylated peptides that have the same ionic and physical properties as the phosphorylated synthetic peptide substrate.
In the present invention, a negative control peptide may include a synthetic peptide identical in sequence to the synthetic peptide substrate with the single exception that the phosphorylated serine or threonine of the amino acid pairs serine-glutamine (SQ), threonine-glutamine (TQ), glutamine-serine (QS), or glutamine-threonine (QT) of the synthetic peptide substrate is replaced with a non-phosphorylated amino acid such as alanine or other neutral amino acid that does not alter the ionic character of the peptide.
In the present invention, a negative control peptide may also include a synthetic peptide identical in amino acid composition and sequence to the synthetic peptide substrate with the single exception that the enhancer amino acid glutamic acid is inserted between the serine and glutamine of the serine-glutamine (SQ) amino acid pair, or between the threonine and glutamine of the threonine-glutamine (TQ) amino acid, or between the glutamine and the serine of the glutamine-serine (QS) pair or between the glutamine and the threonine of the glutamine-threonine (QT) pair of the peptide rather than being located immediately adjacent at the amino- or carboxyl-side of the amino acid pair.
A method of monitoring intracellular protein kinase activity is also provided by the present invention. The method includes introducing an expression vector into a cell, in which the expression vector has the following features to provide monitoring intracellular protein kinase activity: (1) a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for a protein kinase; (2) an optional nuclear localization signal; (3) an optional DNA-binding domain; and (4) a detectable epitope. The expression of this protein substrate provides a phosphorylated protein substrate. By determining the amount of phosphorylated protein substrate, a correlation to the concentration of protein kinase activity in the cell is made.
The present invention also provides a method for monitoring intracellular DNA-PK activity. The method includes introducing an expression vector into a cell, in which the expression vector has the following features to provide for monitoring intracellular DNA-PK activity: (1) a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for DNA-PK; (2) a nuclear localization signal; (3) a DNA-binding domain; and (4) a detectable epitope. The expression of this protein substrate provides a phosphorylated protein substrate. By determining the amount of phosphorylated protein substrate, a correlation to the concentration of DNA-PK activity in the cell is made.
In either embodiment of the present invention for monitoring intracellular protein kinase activity, the protein substrate can further include: (1) an epitope for affinity purification; and (2) a cleavage site which permits excision of the phosphorylation site from the protein segment.
The present invention also provides a composition for monitoring intracellular protein kinase activity. The composition is an expression vector having the following features to facilitate monitoring protein kinase activity: (1) a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for a protein kinase; (2) an optional nuclear localization signal; (3) an optional DNA-binding domain; and (4) a detectable epitope.
A composition for monitoring intracellular DNA-PK activity is also provided by the present invention. The composition is an expression vector having the following features to facilitate monitoring DNA-PK activity: (1) a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for DNA-PK; (2) a nuclear localization signal; (3) a DNA-binding domain; and (4) a detectable epitope.
The present invention also provides a kit for monitoring intracellular protein kinase activity for use in accordance with the methods of the present invention. The kit includes: (1) an expression vector containing a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif, in which the phosphorylation site consensus sequence motif is specific for a protein kinase, a nuclear localization signal, a DNA-binding domain, and a detectable epitope; and (2) means for detecting a phosphorylated protein substrate. The detection of the phosphorylated protein substrate is utilized to determine the amount of protein kinase activity in the cell.
A kit for monitoring intracellular DNA-PK activity for use in accordance with the methods of the present invention is also provided. The kit includes: (1) an expression vector containing a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for DNA-PK, a nuclear localization signal, a DNA-binding domain, and a detectable epitope; and (2) means for detecting a phosphorylated protein substrate. The detection of the phosphorylated protein substrate is utilized to determine the amount of the DNA-PK activity in the cell.
The present invention also provides a method for identifying agents that alter the activity of a protein kinase in a cell. The method includes introducing an expression vector into a cell, in which the expression vector has the following features to facilitate identification of the agents: (1) a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for a protein kinase; (2) an optional nuclear localization signal; (3) an optional DNA-binding domain; and (4) a detectable epitope. The cell is contacted with the suspected agent. The expression of the protein substrate by the cell provides a phosphorylated protein substrate from which the amount of phosphorylated protein substrate is determined. The amount of phosphorylated protein substrate is then correlated to the concentration of protein kinase activity in the cell and changes in the activity resulting from contacting the cell with the suspected agent.
A method for identifying agents that alter the activity of a DNA-PK in a cell is also provided by the present invention. The method includes introducing an expression vector into a cell, in which the expression vector has the following features to facilitate identification of the agents: (1) a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for DNA-PK; (2) a nuclear localization signal; (3) a DNA-binding domain; and (4) a detectable epitope. The cell is contacted with the suspected agent. The expression of the protein substrate by the cell provides a phosphorylated protein substrate from which the amount of phosphorylated protein substrate is determined. The amount of phosphorylated protein substrate is correlated to the concentration of the DNA-PK activity in said cell and changes in the activity resulting from contacting the cell with the suspected agent.
The present invention also provides a method for monitoring a protein kinase activity in a cell through the expression of a reporter gene. The method includes introducing an expression vector into a cell, in which the expression vector has the following features to facilitate monitoring a protein kinase activity: (1) a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for the protein kinase; (2) an optional nuclear localization signal; (3) an optional DNA-binding domain; and (4) a detectable epitope. The expression of the protein substrate provides a phosphorylated protein substrate, which is capable of activating expression of a reporter gene to provide a reporter gene product. By determining the amount of reporter gene product expressed, a correlation to the concentration of protein kinase activity in the cell is made.
A method for monitoring a DNA-PK activity in a cell through the expression of a reporter gene is also provided by the present invention. The method includes introducing an expression vector into a cell, in which the expression vector has the following features to facilitate monitoring DNA-PK activity: (1) a gene coding for a protein substrate having a protein segment containing a phosphorylation site consensus sequence motif specific for DNA-PK; (2) a nuclear localization signal; (3) a DNA-binding domain; and (4) a detectable epitope. The expression of the protein substrate provides a phosphorylated protein substrate, which is capable of activating expression of a reporter gene to provide a reporter gene product. By determining the amount of reporter gene product expressed, a correlation to the concentration of DNA-PK activity in the cell is made.
For a better understanding of the present invention, reference is made to the following description taken together with the accompanying drawings and sequence listings, the scope of which is pointed out in the appended claims.